Purine receptors are involved in the pathophysiology of most disorders of the central
nervous system (CNS) taking part in the early synaptic dysfunction and being in the genesis
and propagation of the inflammatory response in the brain. However, the different ways that
ATP and adenosine receptors contribute to the evolution or to the arrest of deleterious
conditions are still being unraveled.
The broad spectrum of neuroprotection afforded with the blockade of adenosine A2A
receptors under different adverse conditions in the brain can be underlain by a general
mechanism through which these receptors may operate. One possibility is the control of
neuroinflammation, a common event in most CNS disorders. However, while in the peripheral
nervous system A2A receptors have a well established role as a “stop signal” of the
inflammatory cascade, it is not clear whether these receptors trigger or arrest the reactivity of
microglia, the brain immune-competent cells. To tackle this question, it was tested if the
blockade of A2A receptors could prevent biochemical and morphological consequences of
neuroinflammation triggered by the systemic administration of lipopolysaccharide (LPS). In
this work it is shown that the intracerebroventricular injection of a selective A2A receptor
antagonist (SCH58261) was able to prevent the LPS-induced recruitment of activated
microglial cells and the release of the pro-inflammatory cytokine interleukin-1β in the
hippocampus. Moreover, SCH58261 also prevented the LPS-induced activation of mitogenactivated
protein kinases (MAPKs) such as c-Jun N-terminal kinases (JNK) and p38 and the
activation of caspase-3, a key mediator of apoptosis. These results indicate a tight control
mainly of the genesis of neuroinflammation by the blockade of A2A receptors. Therefore, it
was next investigated if A2A receptors were also able to control the direct effects on neurons
of the pro-inflammatory cytokines IL-1β and tumor necrosis factor-α (TNF-α), known to be
important effectors of neuroinflammation-induced deleterious consequences in the
hippocampus. To answer this question, cultured hippocampal neurons were exposed for
different periods to different concentrations of these cytokines and their activation of MAPKs
was evaluated by Western blot and immunocytochemistry. Both TNF-α and IL-1β increased
the phosphorylation (i.e. the activation) of p38 in neurons and IL-1β also increased the
phosphorylation of JNK. In addition, the exposure of hippocampal neurons to IL-1β just
before adding glutamate increased the susceptibility of cells to glutamate-mediated
excitotoxicity. The blockade of A2A receptors with SCH58261 abrogated the activation ofMAPKs induced by IL-1β and prevented the IL-1β-induced exacerbation of excitotoxicity.
Taking advantage of single cell calcium imaging it was found that IL-1β increased both the
calcium entry and the calcium deregulation caused by glutamate exposure in hippocampal
neurons. Pre-incubation of cells with SCH58261 also prevented this effect of IL-1β. Thus, it is
concluded that the antagonism of A2A receptors can control not only the genesis of
neuroinflammation in vivo but also the direct effects of pro-inflammatory cytokines on
neurons, which gives a further insight into the mechanisms operated by these receptors under
pathological conditions.
In addition to adenosine receptors, the ATP (P2) receptors are involved in the
pathophysiology of brain cells in several brain disorders, when the extracellular levels of ATP
are significantly raised. In particular, the pharmacological blockade or the genetic deletion of
P2Y1 receptors conferred a robust neuroprotection against the toxicity induced by the Aβ1-42
peptide (an Alzheimer’s disease related peptide) in hippocampal neuronal cultures and against
Aβ1-42-induced early loss of synaptic markers and mnemonic deficits in rodents. Moreover,
the antagonism of P2Y1 receptors ameliorates the consequences of ischaemic/hypoxic insults
to hippocampal slices. Given the involvement of neuroinflammation in the deleterious effects
of the above conditions, it was hypothesized that the blockade of these receptors could not
only control synaptotoxicity but also neuroinflammation. To begin answering this question it
was investigated whether blocking the P2Y1 receptors could prevent the direct effects of proinflammatory
cytokines on hippocampal neuronal cultures. The results showed that the
selective antagonism of P2Y1 receptors either prevented or attenuated the IL-1β-mediated
effects in hippocampal neurons but, importantly, it prevented the effects of glutamate per se.
Thus it was investigated if the same occurred upon different excitotoxic stimuli to
hippocampal neurons. The results obtained show that neurons are protected against the
toxicity induced by N-methyl-D-aspartic acid (NMDA) or by quinolinic acid when exposed in
the presence of a general P2 receptor antagonist or in the presence of a selective P2Y1
receptor antagonist. This suggests a coupling between P2Y1 receptors and the
neurodegeneration mediated by glutamate NMDA receptors. An impaired function of
glutamate receptors is implicated in the pathophysiology of chronic neurodegenerative
diseases such as epilepsy and in the excitotoxic environment of ischaemia. The previous
results obtained with the antagonism of P2Y1 receptors prompted the testing of the blockade
of these receptors in animal models of temporal lobe epilepsy and stroke.
Epileptic seizures were induced in rats by the intraperitoneal administration of kainate,
which results in a clear neurodegeneration of hippocampal neurons. In this work it is shown that kainate-induced seizures cause a long-term modification of the density of most P2
receptors, in both synaptic and glial membranes, predicting a central role of these receptors in
the aberrant neurotransmission observed in the brain of epileptic animals. In this model, the
blockade of P2Y1 receptors through the intracerebralventricular administration of a selective
antagonist (MRS2500) clearly prevented the early kainate-induced activation of microglia and
the loss of synaptic markers in the hippocampus. Moreover, it also attenuated the seizureinduced
neurodegeneration in hippocampal circuits. Likewise, in a mice model of focal
ischaemia generated by the permanent occlusion of the middle cerebral artery, the general
blockade of P2 receptors or the selective antagonism of P2Y1 receptors prevented both the
ischaemia-induced lesion in the brain and the resultant mnemonic deficits.
Overall, the work presented in this thesis provides evidence supporting the control of
neuroinflammation by the blockade of A2A and of P2Y1 receptors and also the control of
excitotoxicity by the antagonism of P2Y1 receptors in the hippocampus, contributing to the
unraveling of the mechanisms through which these receptors may operate to aggravate
deleterious conditions in the brain